Staphylococcus aureus is a major human pathogen, which can cause serious infections. The organism has become increasingly problematic recently due to emerging multiple-drug resistance strains that are unresponsive to antibiotic treatment. New methods of treatment are therefore urgently needed. Almost all strains of S. aureus produce capsular polysaccharide, which have been classified into 11 setotypes. Type 5 and 8 capsules, which are structurally and genetically similar, are predominant capsules and thus have been used as targets for vaccine development. Recent studies, including our own, have shown that these capsules act as antiphagocytic virulence factors and thereby play an important role in staphylococcal pathogenesis. The produciton of these capsules are influenced by environmental stimuli, however, very little is known about their regulation at the molecular level. Recently, we have characterized the promoter region controlling the expression type 8 capsule genes and have shown that type 5 and 8 capsules are regulated by several known regulators. We have also genetically identified several putative regulatory genes. These results therefore suggest that type 5 and 8 capsules are regulated by a complex regulatory network. In this proposal, we will focus on the regulatory aspect of capsule expression. We propose to accomplish three specific aims: (i) to characterize the regulatory genes involved in the control of capsule production by molecular approaches; (ii) to study the interactions between regulators by genetic and biochemical analyses of mutants deficient in regulatory genes to elucidate regulatory networking; (iii) to investigate the regulation of capsules by environmental stimuli by focusing on carbon dioxide effect both in vitro and in vivo. The successful completion of the studies outlined in this application will undoubtedly unveil the regulatory mechanism controlling the expression of these important virulence factors, which will provide a firm basis for developing new methods of treating staphylocuccal infections.